We propose to form a nationally visible and responsive center focused on fluorescent probe and imaging technologies for investigating pathways and networks in real time and at high resolution in living cells. The proposed Center will be formed by combining the experience and infrastructure of two already existing Centers in Pittsburgh. The research component of the Center will create a powerful toolbox of intracellular fluorescent labels and biosensors that can be used to study many, if not all, the proteins in pathways and networks of living cells. The strong fluorescent probe development program blends genetics, protein structure, nucleic acid structure and fluorescent dye chemistry. The probes we propose to generate will be genetically expressible so that exogenous macromolecules will not have to be transported into the living cells to be studied. Four superb "Driving Biology Projects" are to be part of the overall Center program in addition to the Technology Development Core. These projects are located at the University of Pittsburgh, Carnegie Mellon, Stanford and Berkeley. Their responsibility is to assist the new center in identifying the optimal technologies for development and for providing crucial feedback regarding the utility of the technologies. Imaging and informatics technologies are integrated with the new fluorescent probe technologies so that these combined tools will help cell biologists obtain large amounts of spatial and temporal information about pathways in living cells. Included in the proposal are programs for making the fluorescent probe and imaging technologies robust so that they can be provided to a wide range of cell biologists in academic research, pharmaceutical drug discovery, and biotechnology and to disseminate these technologies and provide training.